Sheeplice, bovicola ovis, allergen treatment

ABSTRACT

The present invention is related to novel nucleotide sequences encoding a louse allergen and a methods for diagnosing, treating and preventing lice infestation and associated allergic disease with the nucleotide sequences and protein allergen of the invention. The present invention also relates to kits for diagnostic assays.

TECHNICAL FIELD

[0001] The present invention concerns novel nucleotide sequencesencoding a louse allergen, particularly although by no means exclusivelyfrom the chewing louse Bovicola ovis, and the use of said nucleotidesequences and protein allergen in the diagnosis, treatment andprevention of lice infestation and associated allergic disease.

BACKGROUND OF THE INVENTION

[0002] Lice are common ectoparasites of mammalian and avian species. Themost important lice in domesticated animals are sucking lice (Insecta:Phthiraptera: Trichodectidae: Anoplura), which have mouthparts able topenetrate the skin of the host and enable the ingestion of tissue fluidsand blood, and the chewing lice, (Insecta: Phthiraptera: Trichodectidae:Mallophaga), which predominantly ingest nutrients from the skin surface,hair, fur, wool or feathers. Chewing lice are common and economicallyimportant particularly on cattle, sheep, goats and equines and are alsofound on dogs, cats and birds including domesticated chickens.

[0003]Bovicola ovis, an example of a chewing louse, is a common externalparasite of sheep world-wide. Infestation of sheep with this parasitehas long been recognised as causing irritation of the skin withconsequent rubbing and damage to the fleece (Johnson, Boray, Plant andBlunt, 1993; Lipson and Bacon-Hall, 1976). Discolouration, reduced yieldand other undesirable qualities may occur in the fleeces of infestedsheep (Kettle and Lukies, 1982; Kettle and Lukies, 1984; Cleland, Dobsonand Meade, 1989). Additionally, recent work by the present inventors hasshown that cockle, a serious defect of lambs' pelts recognised for morethan 100 years (Seymour-Jones, 1913), is also associated withinfestation of sheep with B. ovis (Heath, Cooper, Cole and Bishop, 1995;Heath, Cole, Bishop, Pfeffer, Cooper, and Risdon P, 1995). The inventorshave further shown that cockle is characterized by a superficialperivascular dermatitis with features of an allergic response (Heath,Cole, Bishop, Pfeffer, Cooper, and Risdon P, 1995). Recent studies havesupported the role of allergic immune responses to products of the lousein the development of cockle in sheep (Bany, Pfeffer, Phegan and Heath,1995; Bany, Pfeffer and Phegan, 1995; Pfeffer, Phegan and Bany, 1997;Pfeffer, Bany, Phegan and Osborn, 1993). It can be expected that theallergic response to the louse contributes to the skin irritation thatleads infested sheep to rub and damage their wool and to the skinlesions that severely devalue the pelts from affected lambs.

[0004] The economic consequences of B. ovis infestation of sheep isconsiderable when damage to wool and the costs of prevention ofinfestation are fully accounted (McLeod, 1995). To this can also beadded the substantial cost of reduced quality of lambs pelts due tocockle. Apart from the direct economic costs, the continued use ofconventional treatments to control louse infestation (syntheticinsecticides and insect growth regulators) have detrimental effectsthrough residues entering the environment and food chain as well as onfarmer safety.

[0005] The consumer pressures to reduce the use of such harmfulconventional treatments in the control of louse infestations and thedevelopment of resistance to some synthetic insecticides by licenecessitates refinement of current control strategies and a desire fornew control methodologies and agents.

[0006] It is an object of the present invention to go some way towardsachieving this desideratum or at least provide the public with a usefulchoice.

[0007] The present applicants have identified a louse antigen (allergen)that elicits an allergic response in affected sheep. The identifiedallergen, a protein designated Bo1, has been purified, amino acidsequenced, and the coding cDNA obtained and expressed in the bacterium,Escherichia coli. It is broadly to these allergens and their use indiagnosing, preventing and treating lice infestation and associatedallergic diseases that the present invention is directed.

SUMMARY OF THE INVENTION

[0008] The subject invention concerns the identification, purification,sequencing, and production in recombinant or synthetic form of a novelprotein allergen from chewing lice, including portions of said proteinthat contain at least one B cell or T cell epitope of the protein.

[0009] Accordingly, in one aspect, the present invention may broadly besaid to consist in a substantially purified polypeptide which has theamino acid sequence of SEQ ID NO. 2, or a fragment or a variant thereofhaving substantially equivalent activity.

[0010] According to a further aspect there is provided a polypeptidesubstantially as described above wherein the polypeptide derived from alouse parasitic on an animal provokes a humoral and/or cellularimmunological response in an animal infested by the louse, or a fragmentor a variant thereof having substantially equivalent activity thereto.

[0011] More preferably the variant or fragment incorporates a B cell orT cell epitope of the polypeptide.

[0012] Thus it will be appreciated variants and fragments of thepolypeptide of the invention which may be used to control louseinfestation in animals and associated allergic diseases are alsoincluded in the present invention.

[0013] In general, the animals which may be infested by chewing liceinclude sheep, equines, cattle, dogs, cats or birds including chickens.

[0014] It is to be clearly understood that the invention alsoencompasses peptide analogues, which include but are not limited to thefollowing:

[0015] 1. Compounds in which one or more amino acids is replaced by itscorresponding D-amino acid. The skilled person will be aware thatretro-inverso amino acid sequences can be synthesised by standardmethods; see for example Chorev and Goodman, 1993;

[0016] 2. Peptidomimetic compounds, in which the peptide bond isreplaced by a structure more resistant to metabolic degradation. See forexample Olson et al, 1993; and

[0017] 3. Compounds in which individual amino acids are replaced byanalogous structures for example, gem-diaminoalkyl groups oralkylmalonyl groups, with or without modified termini or alkyl, acyl oramine substitutions to modify their charge.

[0018] The use of such alternative structures can provide significantlylonger half-life in the body, since they are more resistant to breakdownunder physiological conditions.

[0019] Methods for combinatorial synthesis of peptide analogues and forscreening of peptides and peptide analogues are well known in the art(see for example Gallop et al, 1994; Hogan, 1997).

[0020] For the purposes of this specification, the term “peptide andpeptide analogue” includes compounds made up of units which have anamino and carboxy terminus separated in a 1,2, 1,3, 1,4 or largersubstitution pattern. This includes the 20 naturally-occurring or“common” α-amino acids, in either the L or D configuration, thebiosynthetically-available or “uncommon” amino acids not usually foundin proteins, such as 4-hydroxyproline, 5-hydroxylysine, citrulline andornithine; synthetically-derived α-amino acids, such as α-methylalanine,norleucine, norvaline, Cα- and N-alkylated amino acids, homocysteine,and homoserine; and many others as known in the art.

[0021] It also includes compounds that have an amine and carboxylfunctional group separated in a 1,3 or larger substitution pattern, suchas β-alanine, γ-amino butyric acid, Freidinger lactam (Freidinger et al,1982), the bicyclic dipeptide (BTD) (Freidinger et al, 1982; Nagai andSato, 1985), amino-methyl benzoic acid (Smythe and von Itzstein, 1994),and others well known in the art. Statine-like isosteres,hydroxyethylene isosteres, reduced amide bond isosteres, thioamideisosteres, urea isosteres, carbamate isosteres, thioether isosteres,vinyl isosteres and other amide bond isosteres known to the art are alsouseful for the purposes of the invention.

[0022] A “common” amino acid is a L-amino acid selected from the groupconsisting of glycine, leucine, isoleucine, valine, alanine,phenylalanine, tyrosine, tryptophan, aspartate, asparagine, glutamate,glutamine, cysteine, methionine, arginine, lysine, proline, serine,threonine and histidine. These are referred to herein by theirconventional three-letter or one-letter abbreviations.

[0023] An “uncommon” amino acid includes, but is not restricted to, oneselected from the group consisting of D-amino acids, homo-amino acids,N-alkyl amino acids, dehydroamino acids, aromatic amino acids (otherthan phenylalanine, tyrosine and tryptophan), ortho-, meta- orpara-aminobenzoic acid, ornithine, citrulline, norleucine, □-glutamicacid, aminobutyric acid (Abu), and α-α disubstituted amino acids.

[0024] The lice from which the polypeptide is derived belong to thesuborder Mallophaga and preferably from the species Bovicola ovis, achewing louse parasite on sheep.

[0025] Most preferably, the polypeptide comprises the allergendesignated Bo1 from B. ovis.

[0026] Conveniently, the allergen polypeptide of the invention isobtained by expression of a DNA sequence coding therefore in a host cellor organism, or may be chemically synthesised.

[0027] In a further aspect, the present invention provides an isolatednucleic acid molecule encoding a polypeptide substantially as describedabove.

[0028] In a further aspect, the present invention provides an isolatednucleic acid molecule encoding a louse allergen polypeptide of theinvention. Preferably the isolated nucleic acid molecule:

[0029] a) comprises a nucleotide sequence of SEQ ID NO. 1; or

[0030] b) is a functional fragment or variant of the molecule in (a); or

[0031] c) is able to hybridize under stringent conditions to themolecule in (a); or

[0032] d) is the complement of the molecule defined in (a), (b) or (c);or

[0033] e) is an anti-sense sequence corresponding to any of thesequences in (a)-(d).

[0034] This nucleic acid molecule may comprise a DNA, cDNA or RNA.

[0035] Preferably, the fragment or variant of the nucleic acid moleculeabove encodes a B cell or T cell epitope.

[0036] Also provided by the present invention are recombinant expressionvectors which contain a DNA molecule of the invention, and hoststransformed with the vector of the invention capable of expressing apolypeptide of the invention.

[0037] An additional aspect of the present invention provides a ligandthat binds to a polypeptide of the invention. Most usually, the ligandis an antibody or fragment of an antibody containing the binding domain.Most preferably the ligand is a monoclonal or polyclonal antibody whichbinds to the polypeptide of the invention or a functional fragment orvariant thereof. In some other embodiments the ligand may be a phagedisplay molecule.

[0038] In further aspects, the present invention provides a method forassaying samples for the presence of ligands which bind to Bo1 or asegment thereof comprising the steps of obtaining an excretion,secretion, tissue or blood sample from the host and exposing the sampleto a Bo1 ligand binding agent or Bo1 probe via an ELISA or othersuitable assay. When the ligand is an antibody, such assays indicateprior or present infestation by the ectoparasite of the host animal.When the ligand is an antibody of the IgE isotype, such assays areuseful in diagnosis of hypersensitivity to the ectoparasite.

[0039] The present invention also provides a test kit suitable for usein an assay for ligands which bind to Bo1 or a segment thereof whereinthe kit comprises a Bo1 ligand binding agent or probe incorporated intoan ELISA or other suitable assay.

[0040] The present invention also provides an alternative method ofdiagnosing hypersensitivity to the louse in a host (and thus prior orpresent infestation of the host by the louse) via intradermal skintesting. In this method a polypeptide of the invention or fragment orvariant thereof injected intradermally into the host will elicit acharacteristic response in the skin of hypersensitive hosts in contrastto little or no response in non-sensitised hosts. In vitro correlates ofthis method would include exposing isolated tissues or cells of the hostto a polypeptide of the invention as defined above and measuringimmunologically mediated stimulation of the tissues or cells, forexample, release of histamine from blood basophils or proliferation ortransformation of lymphocytes. Use of a polypeptide of the invention asdefined herein would increase the specificity of such methods as to theectoparasite provoking such immunological sensitisation of the hostcompared to published methods where crude antigen preparations were used(Pfeffer, Phegan and Bany (1997;); Bany, Pfeffer and Phegan (1995);Bany, Pfeffer, Phegan and Heath (1995).

[0041] The present invention also provides a vaccine to prevent orreduce Bo1 hypersensitivity in susceptible animals wherein the vaccineincludes an agent selected from the group comprising:

[0042] a) a polypeptide according to the present invention substantiallyas described above;

[0043] b) a nucleic acid molecule according to the present invention;substantially as described above

[0044] c) organisms transfected with and/or expressing the DNA or RNAfor the polypeptide according to (a);

[0045] d) ligands or probes which bind to the polypeptide according to(a).

[0046] The present invention further provides a composition including aneffective amount of an agent selected from the group comprising:

[0047] a) the nucleic acid molecule according to the present inventionsubstantially as described above;

[0048] b) the polypeptide according to the present inventionsubstantially as described above;

[0049] c) organisms transfected with and/or expressing the DNA or RNAfor the polypeptide according to (b); or

[0050] d) ligands or probes which bind to the polypeptide according to(b); together with a pharmaceutically or veterinarily suitable carrieror diluent.

[0051] According to yet a further aspect of the present invention thereis provided a method of diagnosing ectoparasite infestation comprisingthe steps of:

[0052] a) obtaining an excretion, secretion, , tissue or blood samplefrom the host; and

[0053] b) exposing the sample to a ligand or probe for an identifiedantigen present in the ectoparasite's faeces via an ELISA or othersuitable assay.

[0054] The present invention provides test kits for diagnosingectoparasite infestation. In one aspect the test kit may include aligand or probe for an identified antigen present in the ectoparasite'sfaeces incorporated into an ELISA or other suitable assay.

[0055] In preferred embodiments the ectoparasite may be B. ovis and theidentified allergen may be Bo1. Although the above are preferredembodiments they should not be seen as limiting the scope of this aspectof the present invention which may be utilised for a wide range ofectoparasites.

[0056] The invention also encompasses methods of treating animals, orpreventing animals from, exhibiting allergic hypersensitivity to the Bo1polypeptide comprising the step of administering an effective amount ofa vaccine or a composition substantially as described above.

[0057] According to a yet a still further aspect of the presentinvention there is provided a method of diagnosing in an animalhypersensitivity to Bovicola ovis or the Bo1 polypeptide comprising thesteps

[0058] a) injecting intradermally a suitable amount of the polypeptideas claimed in claims 1-8 together with a pharmaceutically orveterinarily suitable carrier or diluent;

[0059] b) at appropriate times thereafter examining the site ofinjection to detect the nature of the reaction to the polypeptide of theinvention

[0060] c) determining on the basis of these observations in comparisonto those on injections of carrier or diluent alone and other controlsolutions whether a specific reaction to the polypeptide of theinvention was evident

[0061] While the invention is broadly as defined above, it will beappreciated by those persons skilled in the art that it is not limitedthereto and that it also includes embodiments of which the followingdescription gives examples.

[0062] Methods and pharmaceutical carriers for preparation ofpharmaceutical compositions are well known in the art, as set out intextbooks such as Remington's Pharmaceutical Sciences, 19th Edition,Mack Publishing Company, Easton, Pa., USA.

[0063] The compounds, vaccines and compositions of the invention may beadministered by any suitable route, and the person skilled in the artwill readily be able to determine the most suitable route and dose forthe condition to be treated. Dosage will be at the discretion of theattendant physician or veterinarian, and will depend on the nature andstate of the condition to be treated, the age and general state ofhealth of the subject to be treated, the route of administration, andany previous treatment which may have been administered.

[0064] The carrier or diluent, and other excipients, will depend on theroute of administration, and again the person skilled in the art willreadily be able to determine the most suitable formulation for eachparticular case.

[0065] For the purposes of this specification it will be clearlyunderstood that the word “comprising” means “including but not limitedto”, and that the word “comprises” has a corresponding meaning.

BRIEF DESCRIPTION OF DRAWINGS

[0066] In particular, preferred aspects of the invention will bedescribed in relation to the accompanying drawings in which:

[0067]FIG. 1 shows a photograph of a silver stained 12% polyacrylamidegel showing protein bands contained in the indicated preparations fromthe louse, Bovicola ovis. Note bands at approximately 28.5, 42 and 83kDa in lane D;

[0068]FIG. 2 shows a photograph of a Western blot of soluble Bovicolaovis antigen reacted with monoclonal antibodies from hybridomas derivedfrom a mouse immunised with soluble Bovicola ovis faecal antigen. Notemajor band at approximately 28.5 kDa and minor bands at approximately 83kDa (lanes 30 to 32, C and D) and at approximately 14 kDa (lanes 27 and28);

[0069]FIG. 3 shows a diagrammatic representation of the strategy used toclone the coding sequence for the mature Bo1 protein into the AY2-4vector;

[0070]FIG. 4 shows a photograph of a Western blot of purified native andrecombinant Bo1 reacted with a Bo1 monoclonal antibody. Note theapparent higher molecular weight of the recombinant compared to thenative Bo1;

[0071]FIG. 5. shows the determination of the cross-reactivity of a Bo1monoclonal antibody with soluble antigens of selected insects and amite;

[0072]FIG. 6. shows the levels of Bo1 antigen detected in wool samplesusing an antigen capture ELISA employing Bo1 monoclonal antibodycompared to louse scores in lambs;

[0073]FIG. 7. shows the skin test results obtained following intradermalinjections of antigens and control solutions in 3 louse-infested (L1,L2, L3) and 3 louse-naive (LF1, LF2, LF3) sheep; and

[0074]FIG. 8. shows the results of an ELISA to detect ovine IgE specificfor crude Bovicola ovis antigen and purified Bo1 protein.

BRIEF DESCRIPTION OF SEQUENCES

[0075] SEQ ID NO. 1 is the nucleotide sequence of the coding DNA of thecomplete Bo1 protein.

[0076] SEQ ID NO. 2 is the amino acid sequence of the complete Bo1protein.

DETAILED DESCRIPTION OF THE INVENTION

[0077] The present applicants have shown for the first time thatBovicola ovis-infested sheep mount immunological responses to theinfesting louse. Evidence of these responses was seen using crudepreparations of soluble allergens of the invention isolated from wholelice and from louse faecal preparations.

[0078] The present invention provides a substantially purified lousepolypeptide allergen which has the amino acid sequence of SEQ ID NO: 2or a fragment or variant thereof having substantially equivalentactivity thereto. Preferably the polypeptide provokes a humoral and/orcellular immunological response in an animal infested by the louse, oris a fragment or variant thereof having substantially equivalentactivity thereto.

[0079] The term “substantially purified” means substantially isolated orseparated away from contaminating proteins or peptides or other materialin the cell or organism in which the polypeptide naturally occurs andincludes polypeptides purified by standard purification techniques aswell as polypeptides prepared by recombinant technology and thosechemically synthesised. Preferably the polypeptide is purified fromwhole lice or lice faecal preparations.

[0080] The term “variant” as used herein refers to nucleotide andpolypeptide sequences wherein the nucleotide or amino acid sequenceexhibits substantially 60% or greater homology with the nucleotide oramino acid sequence of the Figures, preferably 75% homology and mostpreferably 90-95% homology to the sequences of the present invention.—asassessed by GAP or BESTFIT (nucleotides and peptides), or BLASTP(peptides) or BLAST X (nucleotides). The variant may result frommodification of the native nucleotide or amino acid sequence by suchmodifications as insertion, substitution or deletion of one or morenucleotides or amino acids or it may be a naturally-occurring variant.The term “variant” also includes homologous sequences which hybridise tothe sequences of the invention under standard hybridisation conditionsdefined as 2×SSC at 65° C., or preferably under stringent hybridisationconditions defined as 6×SCC at 55° C. Where such a variant is desired,the nucleotide sequence of the native DNA is altered appropriately. Thisalteration can be effected by synthesis of the DNA or by modification ofthe native DNA, for example, by site-specific or cassette mutagenesis.Preferably, where portions of cDNA or genomic DNA require sequencemodifications, site-specific primer directed mutagenesis is employed,using techniques standard in the art.

[0081] The term “ligand” refers to any molecule which may bind toanother molecule such as a polypeptide or peptide and should be taken toinclude, but not be limited to, antibodies and phage display molecules.

[0082] The term “tissue” refers to any coherent collection ofspecialised cells and shall be taken to include, but not be limited to:skin, fur, hair, wool and feathers.

[0083] The reader will appreciate that mimetics of the polypeptides ofthe invention which have substantially identical function as thepolypeptide of the invention are also included within the scope of thepresent invention. The production of such mimetics is within thecapabilities of a skilled worker in the art.

[0084] The polypeptides of the invention can be prepared in a variety ofways. For example, they can be produced by isolation from a naturalsource, by synthesis using any suitable known techniques (such as bystepwise, solid phase, synthesis described by Merryfield (1963), J.Amer. Chem. Soc. Vol 85:2149-2156) or as preferred, through employingDNA techniques.

[0085] The variants of the polypeptides can similarly be made by any ofthose techniques known in the art. For example, variants can be preparedby site-specific mutagenesis of the DNA encoding the native amino acidsequence as described by Adelman et al. DNA 2:183 (1983).

[0086] In addition, polypeptides having substantial identity to theamino acid sequences of the invention can also be employed in preferredembodiments. Here “substantial identity” means that two sequences, whenoptimally aligned such as by the programs GAP or BESTFIT using defaultgap weights, or as measured by computer algorithm BLASTP, share at least60%, preferably 75%, and most preferably 90-95% sequence identity.Preferably, residue positions which are not identical differ byconservative amino acid substitutions. For example, the substitution ofamino acids having similar chemical properties such as charge orpolarity are not likely to affect the properties of a protein. Examplesinclude glutamine for asparagine or glutamic acid for aspartic acid.

[0087] Where it is preferred, recombinant techniques may be used toproduce the polypeptide of the invention, the first step is to obtainDNA encoding the desired product. Such DNA comprises a still furtheraspect of this invention. The DNA of the invention may encode a nativeor modified polypeptide of the invention or an active fragment orvariant thereof.

[0088] Preferably, the DNA comprises an isolated nucleic acid moleculeencoding a louse allergenic polypeptide of the invention, and morepreferably, the nucleic acid molecule comprises the nucleotide sequenceof SEQ ID NO: 1 or a functional fragment or variant thereof.

[0089] The term “isolated” means substantially separated or purifiedaway from contaminating sequences in the cell or organism in which thenucleic acid naturally occurs and includes nucleic acids purified bystandard purification techniques as well as nucleic acids prepared byrecombinant technology, including PCR technology, and those chemicallysynthesised. Preferably, the nucleic acid molecule is derived fromgenomic DNA or the mRNA of the Bovicola ovis chewing louse.

[0090] The DNA can be isolated from any appropriate natural source orcan be produced as intron free cDNA using conventional techniques. DNAcan also be produced in the form of synthetic oligonucleotides where thesize of the active fragments to be produced permits. By way of example,the Triester method of Matteucci et al J. Am. Chem. Soc. Vol103:3185-3191 (1981) may be employed.

[0091] Where desirable, the DNA of the invention can also code for afusion protein comprising the polypeptide of the invention and a carrierprotein. This carrier protein will generally be cleavable from thepolypeptide, fragment or variant thereof under controlled conditions.Examples of commonly employed carrier proteins are βgalactosidase andglutathione-S-transferase.

[0092] As indicated above, also possible are variants of the polypeptidewhich differ from the native amino acid sequence by insertion,substitution or deletion of one or more amino acids. Where such avariant is desired, the nucleotide sequence of the native DNA is alteredappropriately. This alteration can be made through elective synthesis ofthe DNA or by modification of the native DNA by, for example,site-specific or cassette mutagenesis. Preferably, where portions ofcDNA or genomic DNA require sequence modifications, site-specific primerdirected mutagenesis is employed using techniques standard in the art.

[0093] Most preferably, the invention relates to a protein allergen fromBovicola ovis, a chewing louse parasitic on sheep. It will be recognisedby those skilled in the art that nucleotide polymorphism may occur inthe coding DNA and amino acid polymorphism may occur in the protein.Additionally it will be recognised by those skilled in the art that thesame or substantially similar proteins can be expected to occur in otherchewing lice (Suborder Mallophaga). Such proteins can be advantageouslyused in applications as shown for the protein from B. ovis. All suchsequence variations in coding DNA and amino acids of the protein, orportion thereof, are within the scope of the invention.

[0094] In a further aspect, the present invention consists in replicabletransfer vectors suitable for use in preparing a polypeptide or peptideof the invention. These vectors may be constructed according totechniques well known in the art, or may be selected from cloningvectors available in the art.

[0095] The cloning vector may be selected according to the host or hostcell to be used. Useful vectors will generally have the followingcharacteristics:

[0096] (a) the ability to self-replicate;

[0097] (b) the possession of a single target for any particularrestriction endonuclease; and

[0098] (c) desirably, carry genes for a readily selectable marker suchas antibiotic resistance.

[0099] Two major types of vector possessing these characteristics areplasmids and bacterial viruses (bacteriophages or phages). Presentlypreferred vectors include the bacteriophage lambda Uni-ZAP™ XR and themodified plasmid pBAD18 vector, AY2-4 (see FIG. 3 and Guzman, L., Belin,D., Carson, M. J. and Beckwith, J. (1995). Tight regulation, modulation,and high-level expression by vectors containing the arabinose PBADpromoter. J. Bacteriol. 177:4121-4130).

[0100] The DNA molecules of the invention may be expressed by placingthem in operable linkage with suitable control sequences in a replicableexpression vector. Control sequences may include origins of replication,a promoter, enhancer and transcriptional terminator sequences amongstothers. The selection of the control sequence to be included in theexpression vector is dependent on the type of host or host cell intendedto be used for expressing the DNA.

[0101] Generally, eucaryotic, yeast, insect or mammalian cells areuseful hosts. Also included within the term hosts are plasmid vectors.Suitable procaryotic hosts include E. coli, Bacillus species and variousspecies of Pseudomonas. Commonly used promoters such as β-lactamase(penicillinase) and lactose (lac) promoter systems are all well known inthe art. Any available promoter system compatible with the host ofchoice can be used. Vectors used in yeast are also available and wellknown. A suitable example is the 2 micron origin of replication plasmid.

[0102] Similarly, vectors for use in mammalian cells are also wellknown. Such vectors include well known derivatives of SV-40, adenovirus,retrovirus-derived DNA sequences, Herpes simplex viruses, and vectorsderived from a combination of plasmid and phage DNA.

[0103] Further eucaryotic expression vectors are known in the art (e.g.P. J. Southern and P. Berg, J. Mol. Appl. Genet. 1327-341 (1982); S.Subramani et al., Mol. Cell. Biol. 1, 854-864 (1981); R J. Kaufmann andP. A. Sharp, “Amplification and Expression of Sequences Cotransfectedwith a Modular Dihydrofolate Reducase Complementary DNA Gene, J. Mol.Biol. 159, 601-621 (1982); R J. Kaufmann and P. A. Sharp, Mol. Cell.Biol. 159, 601-664 (1982); S. I. Scahill et al., “Expressions AndCharacterization Of The Product Of A Human Immune Interferon DNA Gene InChinese Hamster Ovary Cells,” Proc. Natl. Acad. Sci. USA. 80, 4654-4659(1983); G. Urlaub and L. A. Chasin, Proc. Natl. Acad. Sci. USA. 77,4216-4220, (1980).

[0104] The expression vectors useful in the present invention contain atleast one expression control sequence that is operatively linked to theDNA sequence or fragment to be expressed. The control sequence isinserted in the vector in order to control and to regulate theexpression of the cloned DNA sequence. Examples of useful expressioncontrol sequences are the lac system, the trp system, the tac system,the trc system, major operator and promoter regions of phage lambda, theglycolytic promoters of yeast acid phosphatase, e.g. Pho5, the promotersof the yeast alpha-mating factors, and promoters derived from polyoma,adenovirus, retrovirus, and simian virus, e.g. the early and latepromoters of SV40, and other sequences known to control the expressionof genes of prokaryotic and eucaryotic cells and their viruses orcombinations thereof.

[0105] A preferred promoter for use herein is the Arabinose promotor(Guzman, L., Belin, D., Carson, M. J. and Beckwith, J.,1995. ), however,any suitable promoter is included within the scope of the presentinvention as would be appreciated by a skilled worker.

[0106] In the construction of a vector it is also an advantage to beable to distinguish the vector incorporating the foreign DNA fromunmodified vectors by a convenient and rapid assay. Reporter systemsuseful in such assays include reporter genes, and other detectablelabels which produce measurable colour changes, antibiotic resistanceand the like. In one preferred vector, the β-galactosidase reporter geneis used, which gene is detectable by clones exhibiting a blue phenotypeon X-gal plates. This facilitates selection. In one embodiment, theβ-galactosidase gene may be replaced by a polyhedrin-encoding gene;which gene is detectable by clones exhibiting a white phenotype whenstained with X-gal. This blue-white color selection can serve as auseful marker for detecting recombinant vectors.

[0107] Once selected, the vectors may be isolated from the culture usingroutine procedures such as freeze-thaw extraction followed bypurification.

[0108] For expression, vectors containing the DNA of the invention to beexpressed and control signals are inserted or transformed into a host orhost cell. Some useful expression host cells include well-knownprokaryotic and eucaryotic cells. Some suitable prokaryotic hostsinclude, for example, E. coli, such as E. coli S G-936, E. coli HB 101,E. coli W3110, E. coli X1776, E. coli X2282, E. coli DHT, and E. coli,MR01, Pseudomonas, Bacillus, such as Bacillus subtilis, andStreptomyces. Suitable eucaryotic cells include yeast and other fungi,insect, animal cells, such as COS cells and CHO cells, human cells andplant cells in tissue culture.

[0109] Depending on the host used, transformation is performed accordingto standard techniques appropriate to such cells. For prokaryotes orother cells that contain substantial cell walls, the calcium treatmentprocess (Cohen, S N Proc Nat Acad Sci, USA 69 2110 (1972)) may beemployed. For mammalian cells without such cell walls the calciumphosphate precipitation method of Graeme and Van Der Eb, Virology 52:546(1978) is preferred. Transformations into plants may be carried outusing Agrobacterium tumefaciens (Shaw et al., Gene 23:315 (1983) or intoyeast according to the method of Van Solingen et al. J. Bact. 130: 946(1977) and Hsiao et al. Proc Nat Acad Sci, USA 76:3829 (1979).

[0110] Upon transformation of the selected host with an appropriatevector the polypeptide encoded can be produced, often in the form offusion protein, by culturing the host cells. The polypeptide of theinvention may be detected by rapid assays as indicated above. Thepolypeptide is then recovered and purified as necessary. Recovery andpurification can be achieved using any of those procedures known in theart, for example by absorption onto and elution from an anion exchangeresin. This method of producing a polypeptide of the inventionconstitutes a further aspect of the present invention.

[0111] Host cells transformed with the vectors of the invention alsoform a further aspect of the present invention.

[0112] In a further aspect, the present invention provides a ligand thatbinds to a polypeptide of the invention.

[0113] In one embodiment the ligand may be an antibody or antibodyfragment raised against the polypeptide of the invention. Suchantibodies may be polyclonal, but are preferably monoclonal.

[0114] Polyclonal antibodies may be produced according to the methodused by Koelle el al.; Cell 67:59-77, 1991 incorporated herein byreference. Useful antibody production protocols are outlined in U.S.Pat. No. 5,514,578 incorporated herein by reference. Monoclonalantibodies may be produced by methods known in the art. These methodsinclude the immunological method described by Kohler and Milstein inNature 256:495-497 (1975) as well as by the recombinant DNA methoddescribed by Huse et al. Science 246:1275-1281 (1989). Any of the assaymethods detailed in U.S. Pat. No. 5,514,578 are also incorporated foruse herein by reference.

[0115] An understanding of the tertiary structure and spatialinteractions between the Bo1 allergen (especially ligand-bindingdomains) and its ligand will provide ways to select highly specificligands which may be bound only by a modification of a natural receptorligand-binding domain. Also, this knowledge will provide directions fornew designs using the combination of Bo1 allergens with ligands andmethods to design and select peptide mimetics of ligands with highspecificity by techniques such as phage differential display.

[0116] In another embodiment the ligand may comprise molecules that bindto the polypeptide of the invention which are derived from naturalsources, including plants, animals and insects. Insect extracts whichproduce mimetics of the Bo1 allergen are of particular interest.

[0117] Accordingly, in a further aspect, the present invention providesa method of assaying samples for the presence of ligands. Assayingprocesses using polypeptides as a ligand binding agent or probe are wellwithin the capacity of the art skilled worker. The selection of thesegment to be used as a probe will allow particular functionallyassociated segments to be isolated. For example, if a segment of thepolypeptide binding domain of the present invention is used as a probe,identical or similar polypeptide binding domains can be identified,isolated and the encoding DNA determined.

[0118] It will also be appreciated that the selection of probes highlyspecific for Bovicola ovis, will provide an opportunity to assay samplesin a rapid and highly specific manner to detect the presence of Bovicolaovis.

[0119] Samples of material to be screened may be prepared in the form ofsubstrate solutions, then exposed to the ligand binding agent or probe.The presence of a ligand binding agent/ligand complex may be detectedaccording to methods also known in the art. Examples of such methodsinclude agglutination, radioimmunoassay, fluorescence or enzymeimmunoassay techniques. A suitable screening test is an ELISA assay. Inthis method of the invention it is presently preferred that the Bo1binding domain be used as the ligand binding agent.

[0120] In a further aspect the present invention provides test kitssuitable for use in such assays. An example of such a test kit is anELISA assay test kit including a ligand binding agent of the invention.

[0121] In a further aspect, the present invention provides a method ofassaying samples for the presence of polypeptides or fragments orvariants thereof or other antigenic molecules excreted in the faeces ofectoparasites that are specific for individual species or related groupsof ectoparasites. Examples of such methods include agglutination,radioimmunoassay, fluorescence or enzyme immunoassay techniques. Asuitable screening test is an ELISA assay including a ligand (orligands) that binds to an identified antigenic molecule in the faeces ofthe ectoparasite. Such assays will enable convenient and rapid screeningof multiple samples from hosts for the detection of infestation of thehost by ectoparasites.

[0122] In this method of the invention it is presently preferred thatmonoclonal antibody be used as the ligand to detect ectoparasiteinfestation.

[0123] In a further aspect the present invention provides test kitssuitable for use in such assays. An example of such a test kit is anELISA assay test kit including a ligand of the invention.

[0124] A further aspect of the present invention provides a method ofdiagnosing hypersensitivity to the louse in a host (and thus prior orpresent infestation of the host by the louse) via intradermal skintesting. In this method a polypeptide of the invention or fragment orvariant thereof injected intradermally into the host will elicit acharacteristic specific response in the skin of hypersensitive hosts incontrast to little or no response in non-sensitised hosts. The responsein the hypersensitive host will include a least one of the followingresponses at the site of injection; wheal, flare, induration. This useof intradermal skin testing employing injection of an allergenpreparation together with injection of a negative control preparationand histamine at adjacent skin sites is well known to those skilled inthe art. In vitro correlates of this method will also be appreciated bythose skilled in the art and include exposing isolated tissues or cellsof the host to a polypeptide of the invention as defined above andmeasuring immunologically mediated stimulation of the tissues or cells,for example, release of histamine from blood basophils or proliferationor transformation of lymphocytes. Use of a polypeptide of the inventionas defined herein would increase the specificity of such methods forimmunological sensitisation of the host by Bovicola ovis compared topublished methods where crude antigen preparations were used (Pfeffer,Phegan and Bany (1997;); Bany, Pfeffer and Phegan (1995); Bany, Pfeffer,Phegan and Heath (1995).

[0125] Diagnostic assays or tests employing the protein, peptides and/orspecific antibodies or synthetic molecules that mimic these embodimentsare considered to be part of this invention and may be useful firstly,to identify infestation with the chewing lice in animals, and secondly,to identify hypersensitivity in infested animals in vivo or in vitro.

[0126] Further it will be obvious to those skilled in the art, that thesubject polypeptides, peptides and antibodies or other molecules thatspecifically bind or mimic the subject protein and peptides andantibodies may be used as novel agents to control infestation by chewinglice or to prevent or suppress the immunological hypersensitivityarising as a consequence of such infestation and are, as such, includedin the scope of the present invention. Firstly, the protein or peptidesin native form or modified, the total coding DNA of the protein or partthereof, recombinants incorporating all or part of the protein,organisms transfected with and/or expressing the coding DNA or RNA forthe protein or peptides, and synthetic molecules that copy or mimic theprotein or peptides may be formulated into vaccines to elicit protectiveimmunity in the host to the chewing louse. Further, antibodies specificto epitopes of the protein or peptides of the invention, or syntheticmolecules mimicing these antibodies may be used to passively immunizethe host so that the host is partially or completely protected frominfestation with the louse. Secondly, the protein or peptides in nativeor modified forms, organisms transfected with and/or expressing thecoding DNA or RNA for the protein or peptides, or specific monoclonal orpolyclonal antibodies or synthetic molecules that mimic the specificantibodies may be used to damage the chewing lice or interfer withphysiological processes of the chewing lice. Thirdly, it will be obviousthat the protein or peptides in native form or modified, the totalcoding DNA of the protein or part thereof, and recombinantsincorporating the protein or peptides, organisms transfected with and/orexpressing the coding DNA or RNA for the protein or peptides, andsynthetic molecules that mimic the protein, peptides or specificantibodies may be formulated into treatments to prevent, ameliorate orreverse the allergic hypersensitivity that develops in the host animalin response to infestation by the chewing louse. It is intended thatthese applications also be included in this patent.

[0127] The protein or peptides of the invention may be formulated intovaccines which when administered to animals may elicit a protectiveresponse against the louse. Alternatively, a polynucleotide molecule ofthe invention may be incorporated into a vector or plasmid ortransformed into a host which when administered to the animal may alsoelicit a protective response against the louse. It is also possible thatthe antibodies, fragments of antibodies, phage display molecules, ortransformed hosts containing or secreting such molecules may besystemically administered to an animal and thereby provide passiveprotection. The Bo1 protein may be required for functions important forthe viability or fecundity of the louse and thus the invention may beused to interfere with this function and thereby prevent or controlinfestations by the louse. Synthetic or recombinant molecules whichblock the function of the protein or disrupt regulation of production ofthe protein in the louse may be designed from knowledge of the sequencesof the invention, synthetised and advantageously applied to animals. Thehypersensitivity elicited by infestations of the louse may be preventedor reduced by administering the protein, peptides, polynucleotidemolecules or transformed hosts of the invention to susceptible animalsin regimens that prevent the development of or downregulate theimmunological responses leading to hypersensitivity disease. Examples ofsuch regimens may include variations of route of administration andcoadministration with various adjuvants, cytokines or organisms. Thepresent invention may also be used to define B and T cell epitopes ofthe Bo1 protein important in the hypersensitivity elicited in the hostanimal. This may be done by synthetising overlapping peptides anddetermining recognition of the individual peptides by antibodies or Tcells from hypersensitive hosts. Such defined epitopes may be used inpreventing or controlling the hypersensitivity. For example, peptidescontaining epitopes of the protein of the invention may be used indesensitisation regimens of animal hosts without the danger ofcross-linking IgE on mast cells and thereby eliciting anaphylaxis. Theuses described herein are intended to be encompassed by the presentinvention.

[0128] Non-limiting examples illustrating the invention will now beprovided.

[0129] It will be appreciated that the above description is provided byway of example only and variations in both the materials and techniquesused which are known to those persons skilled in the art arecontemplated.

PROTOCOL EXAMPLE 1 Preparation of Soluble Antigen from Whole Lice andLouse Faeces

[0130] Live nymph and adult Bovicola ovis were collected from infestedsheep and separated from wool and any other debris in a glass petri dishby raising one side of the dish and allowing the lice to migrate to thelower side. The lice were then placed in a ceramic mortar and snapfrozen by adding liquid nitrogen. While maintaining the mortar overliquid nitrogen, the lice were crushed to a fine powder with a pestle.The powder was then allowed to thaw briefly and cold phosphate bufferedsaline containing 1 mM Pefabloc® (Boehringer Mannheim) was added at therate of 10 ml per gram of lice. The preparation was transferred to aglass homogeniser maintained over ice and homogenised. The preparationwas ultracentrifuged (10 000 g, 20 min, 4° C.) to remove particulatematter. The supernatant containing the soluble antigen was filteredthrough sterile 0.2 μm filters. For short-term storage, the supernatantwas held at 4° C. For long-term storage, the supernatant was mixed 1 to1 by volume with glycerol (AnalaR®, BDH) and stored at −20° C. Typicallythe protein concentrations of the supernatants following mixing withglycerol were 2 to 3 mg per ml when measured using the BCA Protein Assay(Pierce) after precipitation with 5% trichloroacetic acid. The complexnature of the crude soluble antigen prepared from whole lice is shown inFIG. 1.

[0131] To obtain louse faeces, lice separated from wool and debris weremaintained overnight in clean glass petri dishes under conditions ofcontrolled temperature and relative humidity according to the method ofHoplins, 1970. In vitro colonization of the sheep biting louse, Bovicolaovis. (Annals of the Entomological Society of America. 63:1196-1197).Lice were then poured off the plate and any dead lice, parts of lice orother debris attached to the surface of the plate removed. The faecalpellets attached to the glass plate were suspended in 10 ml phosphatebuffered saline containing 1 mM Pefabloc® and transferred to a glasshomogeniser maintained over ice and homogenised. This preparation wasthen ultracentrifuged and filtered as above and stored at 4° C. Proteinlevels in soluble louse faecal antigen preparations were measured byabsorbance at 280 nm or by the BCA Protein Assay (Pierce).

EXAMPLE 2 Isolation of Ovine IgE and Coupling to Affinity Columns

[0132] Sera collected from sheep infested with B. ovis were screened byELISA to identify those with higher levels of IgE binding with wholelouse soluble antigen. A selected serum was then diluted 1 to 5 byvolume with wash buffer (50 mM phosphate buffer, 500 mM NaCl buffer, pH7.0) and filtered through a 0.2 μm filter. The IgE in the diluted serumwas separated using an immunoaffinity column constructed by coupling amonoclonal antibody specific for ovine IgE to a HiTrap NHS-activatedcolumn (Pharmacia Biotech) as described by Shaw, R. J., Grimmett, D. J.,Donaghy, M. J., Gatehouse, T. K., Shirer, C. L. and Douch, P. G. C.1996. Production and characterisation of monoclonal antibodiesrecognising ovine IgE. Veterinary Immunology and Immunopathology.51:235-251. The eluates from the IgE specific affinity column weredialysed against wash buffer and further purified by passing thepreparation over affinity columns to which were coupled an irrelevantmonoclonal antibody or protein G. Analysis of the resultant preparationsby SDS PAGE under reducing conditions showed bands typical of IgE heavyand light chain at high (>90%) levels of purity. Approximately 10 mg ofovine IgE were coupled to 1 ml HiTrap NHS-activated columns (PharmaciaBiotech) as per the manufacturer's recommendations.

EXAMPLE 3 Isolation of Native Allergens from Bovicola ovis UsingOvine-IgE Immunoaffinity Chromatography

[0133] Crude soluble B. ovis and B. ovis faecal antigen preparationswere prepared as in Example 1 except that the diluent was wash buffer(50 mM phosphate buffer, 500 mM NaCl buffer, pH 7.0). The dilutedantigen preparations were loaded onto an ovine IgE immunoaffinity columnconstructed as in Example 2. The column was then washed with washbuffer, and the native allergens were eluted with 100 mM glycine at pH3.0. The eluates were returned to neutral pH by adding 1 M Tris, 1.5 MNaCl, pH 8.0 at a ratio of 1 to 10 (v/v). The allergen eluates wereconcentrated by ultrafiltration (MicrosepTM Centrifugal Concentrators,Pall Filtron Corporation, cutoff 3000 kD) and examined by SDS-PAGE underreducing conditions. Intradermal skin testing confirmed that the eluatesfrom the whole louse and the louse faeces antigen preparations containedallergens.

EXAMPLE 4 Preparation of Monoclonal Antibody

[0134] BALB/c mice were injected subcutaneously with louse faecalantigen preparation (up to 1 mg total protein) mixed 1 to 1 withFreund's Complete Adjuvant and boosted intraperitoneally with similaramounts of louse faecal antigen in Freund's Incomplete Adjuvant on 2occasions. Mice showing robust antibody responses were identified byassaying serum samples from the mice in ELISA for reactivity with lousefaecal antigen. Spleen lymphocytes from selected mice were fused withNS-1 myeloma cells by standard techniques. The resultant hybridomas wereplated out in 1 ml cultures over 5, 24 well plates in selective mediaconditioned with BALB/c thymocytes. Subsequently, the media in the wellswas screened by ELISA for murine IgG antibody recognizing soluble faecalantigen. The hybridomas from positive wells were subjected to limitingdilution in 96 well culture plates at mean concentrations of 0.5, 1 and2 cells per well. These hybridomas were again screened for antibodyrecognizing crude soluble louse faecal antigen. Clones producingantibody to faecal antigen were also screened by ELISA to determinereactivity of the antibody with isolated native allergens prepared asdescribed in Example 3. Single hybridomas identified as producingantibody to native allergens were cloned a second time by limitingdilution. Hybridomas from the second cloning were expanded andcryopreserved. Monoclonal antibodies selected for further use were ofthe murine IgG-1 isotype. For the production of monoconal antibody,cloned hybridomas were expanded by standard culture techniques, allowedto overgrow and the supernatant collected.

[0135] Monoclonal antibodies produced by the hybridomas recognised animmunodominant molecule with a major moiety of 28.5 kDa rMW on Westernblots of crude louse or louse faecal antigen (FIG. 2). This correspondedto one of the major bands observed on SDS PAGE of putative nativeallergens obtained by IgE affinity chromatography of both whole louseand louse faecal antigen preparations. Minor bands were also observed atapproximately 14, 42, and 83 kDa rMW. The higher rMW moieties appear torepresent multiples of the 14 kDa band, that is, 2×14=28 (˜28.5),3×14=42 and 6×14=84 (˜83).

[0136] The protein identified by the monoclonal antibodies wasdesignated Bo1.

EXAMPLE 5 Purification of Bo1 Allergen

[0137] Monoclonal antibody prepared as described in Example 4 waspurified over a Protein G affinity chromatography column and coupled toa HiTrap NHS-activated column (Pharmacia Biotech) in a similar manner tothat described in Example 2. The column was used to obtain eluates fromsoluble whole louse antigen prepared as described in Example 1.

[0138] The eluates from the monoclonal antibody affinity columncontained a protein at high levels of purity and with characteristicsconsistent with the protein observed in crude louse antigen by SDS PAGE(FIG. 1) and Western blots probed with the monoclonal antibody (FIG. 2).Additionally, the purified allergen was recognised by IgE fromlouse-infested sheep on Western blots and in ELISA (FIG. 8). Intradermalskin testing confirmed specific responsiveness to Bo1 in louse-infestedlambs compared to louse-naive lambs (FIG. 7).

EXAMPLE 6 Amino Acid Sequencing of the Bo1 Allergen

[0139] Native Bo1 purified using the monoclonal antibody affinity columnwas subjected to SDS PAGE under reducing conditions and electroblottedto PVDF membrane (Problott™, Applied Biosystems) using standardtechniques for preparing proteins for sequencing. The PVDF membrane wasstained with 0.1% Ponceau S and the 28.5 kDa band was identified and cutout. The membrane pieces were briefly washed with methyl alcoholcontaining 0.1% triethylamine followed by 2 washes with methyl alcoholalone. Automated microsequencing was carried out on a gas phaseinstrument (470AR/120A/920A/610A, Applied Biosystems). The N-terminalamino acid sequence obtained was (a) SPTELDLRLLVETARDISVILFKNLHAGYN

[0140] The Bo1 28.5 kDa band was also cut from gels following SDS PAGEunder reducing conditions for in-gel trypsin digests. The in-gel trypticdigestion followed the protocol of Rosenfeld, J., Capdevielle, J.,Guillemot, J. C. and Ferrara, P. (1992) In-gel digestion of proteins forinternal sequence analysis after one- or two-dimensional gelelectrophoresis, Analytical Biochemistry 203:173-179. Peptides elutedfrom the gel were then separated on a Phenomenex Jupiter C18 column (300angstrom, 5 micron, 2×250 mm) attached to a microbore HPLC (PEBiosystems, 140A delivery system and 1000S Diode array detector).Selected peptides were sequenced on a PE Biosytems Procise proteinsequencer (model 492) using chemicals and methods supplied by themanufacturer. The following sequences were obtained (b) DISVILFK (c)NLHAGYNEVNPK (d) VFTNIK (e) IGEQVLK (f) (I)NVIFK (g) KLFDTEVPEVVK (h)DISVTLFK (i) IEILLNELAPEAK (j) TLIGALDQ(L)K

EXAMPLE 7 RNA Isolation and cDNA Library Construction

[0141] RNA was isolated from B. ovis essentially as described by FrenkelM. J., Savin K. W., Bakker R. E., and Ward C. W. (1989).Characterization of cDNA clones coding for muscle tropomyosin of thenematode Trichostronglus colubriformis, Molecular and BiochemicalParasitology. 37:191-200. B. ovis (100 mg) snap frozen in liquidnitrogen were ground with a pestle and mortar over liquid nitrogen. Oneml of 6 M Guanidine-HCL, 0.2 M sodium acetate (pH 5.2) plus 10 mMβ-mercaptoethanol was added, ground with the B. ovis and the powdertransferred to an eppendorf tube. Two hundred μl of 95% ethanol wasadded and the mixture placed on dry ice/ethanol for 5 minutes. Themixture was centrifuged for 5 minutes at 4° C. and the pellet thenresuspended in 500 μl of 6 M Guanidine-HCL, 0.2 M sodium acetate (pH5.2) plus 10 mM EDTA. Ethanol precipitation and centrifugation wasrepeated and the pellet resuspended in 250 μl of urea buffer (7M urea,100 mM Tris-HCL (pH 7.5), 0.1 mM EDTA, 0.1% (w/v) SDS and then 500 μl ofwater saturated phenol:chloroform (1:1) was added. After centrifugationfor 10 mins at 4° C. the aqueous layer was transferred to a new tube andthe RNA was ethanol precipitated, dried and resuspended in 50 μl ofdouble distilled water.

[0142] A cDNA library was synthesized from the B. ovis mRNA using aZAP-cDNA® Synthesis Kit (Stratagene). The cDNA was ligated into thebacteriophage lambda Uni-ZAPTM XR vector arms with T4 DNA ligase andpackaged with GIGAPACK® II Packaging Extract.

EXAMPLE 8 Cloning and Characterisation of the Complete Coding DNA forBo1

[0143] Based on the Bo1 amino acid sequences, an oligonucleotide primer(BoP14-A) was designed to hybridise to Bo1 cDNA encoding the aminoterminal region (amino acid sequences (a), (b), (c) and (h) in Example6) and a second oligonucleotide (BoP14-B) was designed to hybridise toBo1 cDNA encoding an internal peptide (amino acid sequence (g) inExample 6). The oligonucleotides were designed to hybridise to opposingstrands of the cDNA such that they would amplify the intervening cDNAwhen used as primers in a polymerase chain reaction (PCR) with cDNAderived from B. ovis mRNA. BoP14-A CATGCTGGATATAATGAAGT(A/T)AA(C/T)CCBoP14-B TTAACAACTTCAGGAACTTC(A/T)GT(A/G)TC(A/G)AA

[0144] The conditions for the PCR reaction were primers at 0.3 μM, dNTPsat 200 μM and 20 ng of template, 3 cycles of 94° C. for 30 sec, 45° C.for 60 sec and 72° C. for 60 sec; 30 cycles of 94° C. for 30 sec, 50° C.for 60 sec and 72° C. for 60 sec; and then held at 72° for 10 min.

[0145] Following PCR, an amplified DNA fragment of approximately 390 bpwas identified by agarose gel electrophoresis. This fragment waspurified using the Wizard® PCR Preps DNA Purification System (Promega)and then radiolabeled with [α-³²P]dCTP using the RTS RadPrime DNALabelling System (Life Technologies). The radiolabeled Bo1 cDNA probewas used to screen the B. ovis cDNA library to identify DNA cloneshomologous to the Bo1 cDNA essentially as described in Sambrook, J.,Fritsch, E. F. and Maniatis T. (1989). Molecular cloning: a laboratorymanual, 2nd ed. New York (Cold Spring Harbour Laboratory Press).Approximately 45 000 phage clones from the library were plated onto agarplates with E. coli XL1-Blue MRF’ cells and incubated to produce phageplaques. The plaques were replicated onto Hybond-N+ nylon membranes(Amersham) and processed for hybridisation with the probe. Positivehybridising clones were identified and these were plaque purified untilthey were clonal. The B. ovis cDNA inserts were amplified by PCR usingprimers that hybridised to the DNA surrounding the cDNA cloning site andthe PCR products were sequenced. From the sequences, the complete codingsequence of the Bo1 allergen could be discerned (SEQ. ID No. 1). Thesequence predicts a protein of approximately 30 kDa having an aminoterminal secretory leader sequence. From the nucleotide sequence, theamino acid sequence (SEQ. ID No. 2) was deduced using codon preferencesrecognised for B. ovis and the N-terminal and internal sequencesdetermined in Example 6.

EXAMPLE 9 Expression of Recombinant Bo1 Protein

[0146] The coding cDNA for the mature Bo1 protein, as predicted from theN-terminal amino acid sequence (Example 6, amino acid sequence (a)), wasamplified by PCR from a B. ovis cDNA template. The oligonucleotideprimers used in the PCR reaction to amplify the Bo1 cDNA were designedfrom the cDNA sequence obtained above (SEQ. ID. No. 1). The primers wereBo1-X2 CTTGCGGCCGCCATTTTTGCAACACAGTCTG Bo1-X3CGCGGATCCATATGTCCCCAACAGAACTCGAT

[0147] Primer Bo1-X2 was designed to be homologous to the Bo1 cDNAencoding the amino terminus of the mature protein as identified by aminoacid sequencing of the intact, purified Bo1 protein (amino acidsequences (a) in Example 6). Primer Bo1-X3 was designed to be homologousto the carboxyl terminal Bo1 amino coding DNA (SEQ. ID. No. 1). Theprimers also contained restriction enzyme cleavage sites to permit theligation of the amplified DNA into an expression vector. Following astandard PCR reaction (35 cycles of 95° C. for 30 sec, 50° C. for 30sec, and 72° C. for 1 min) with Bo1-X2 and Bo1-X3 primers and the Bo1cDNA template, the product of approximately 700 bp was resolved byagarose gel electrophoresis. This PCR fragment was digested by NdeI andNotI endonucleases and cloned into the AY2-4 vector, a derivative ofpBAD18 (Guzman, L., Belin, D., Carson, M. J. and Beckwith, J. (1995).Tight regulation, modulation, and high-level expression by vectorscontaining the arabinose PBAD promoter. J. Bacteriol. 177:4121-4130. ),resulting in the Bo1 expression vector shown in FIG. 3. Joining of theamplified coding DNA to the expression vector resulted in the fusion ofthe complete mature Bo1 coding sequence to the initiator methioninecodon of the expression vector at a Nde1 restriction enzyme cleavagesite. The carboxyl coding end of the Bo1 cDNA was joined at a Not1 siteto vector DNA in frame with DNA encoding the E-tag epitope (Pharmacia)and the nonapeptide AAAHHHHH followed by a termination codon. Theexpression vector drives expression of the ligated coding DNA from thearabinose PBAD promoter and thus when electroporated into E. coli XL2,recombinant Bo1 is produced in response to exogenous arabinose in thegrowth medium (Guzman, L., Belin, D., Carson, M. J. and Beckwith, J.(1995). The recombinant Bo1 produced in this way has a carboxyl terminalfusion that includes the E-tag epitope and the nonpeptide AAAHHHHHH.

[0148] The transformed E. coli were induced to express the recombinantBo1 by the addition of 0.2% L(+)arabinose to the culture medium(Luria-Bertani medium, Sigma, with Ampicillin, Sigma, at 100μg/ml). Theexpressed recombinant protein was produced in the bacterial cytosol andwas extracted by sonication of the bacteria and centrifugation to removeinsoluble material. The recombinant Bo1 (rBo1) was then purified byimmobilised metal affinity chromatography exploiting the affinity of thehexahistidine tag at the carboxyl terminus for immobilised nickel(HiTrapTM Chelating column, Amersham Pharmacia Biotech AB) and elutedwith immadazol medium of increasing concentrations. Some preparations ofrBo1 were further purified over the Bo1 mAb affinity column (Example 5).Because the recombinant protein has the carboxyl terminal fusionpartner, it has a higher rMW (approximately 29.5 kDa) than the nativeprotein on SDS PAGE under reducing conditions (FIG. 4).

[0149] The rBo1 was recognised by the Bo1 mAb on western blots (FIG. 4)and in ELISA. In preliminary trials, rBo1 was also recognisedpreferentially in ELISA by IgE from louse-infested sheep compared tolouse-naive sheep.

EXAMPLE 10 The Specificity of Bo1 mAb for B. ovis

[0150] The specificity of Bo1 mAb for B. ovis was examined in ELISA bydetermining its reactivity with soluble antigen preparations made from arepresentative variety of insects to which sheep may be exposed in NewZealand and a mite. The antigens were prepared from adult sandflies,adult mosquitoes, and maggots of blowflies by crushing the insects inglass homogenisers in cold PBS containing 1 mM Pefabloc® (BoehringerMannheim). Antigen preparations were clarified by centrifugation. B.ovis antigen was prepared as described in Example 1. D. pteronyssinusantigen was obtained commercially (Allergenic Extract, Standardized MiteDP, Bayer Corporation). After suitable dilution, the antigens were usedto coat wells in microtitre plates and the ELISA performed using the Bo1mAB as the primary antibody, and goat anti-mouse IgG conjugate as thesecond antibody using standard protocols. Bo1 mAb showed substantialreactivity only with antigen prepared from B. ovis (FIG. 5).

EXAMPLE 11 Use of B. ovis-specific Antibody in an in vitro DiagnosticAssay to Detect Louse Infestation

[0151] An antigen capture ELISA was used to test for the presence of Bo1on the wool of louse-infested and louse-naive lambs as shown in thefollowing example.

[0152] Twenty-nine louse-infested and 12 louse-naive lambs, maintainedat pasture, were scored for levels of louse infestation by counting thetotal number of lice observed in 10 cm long wool partings at 12predetermined sites over the body. Wool samples were cut at skin levelfrom the mid-shoulder region of these lambs and placed in individualpaper bags and stored at room temperature. One gram of wool from eachsample was placed in a glass container and mixed with 20 ml of buffer(PBS plus 0.5% Tween 20) for 2 hours at room temperature. Thesupernatants were decanted and used in the following antigen captureELISA.

[0153] Monoclonal antibody specific for Bo1 (Bo1 mAb) was purified fromhybridoma supernatants over a Protein G affinity chromatography column(Pharmacia) and concentrated using a 30 kDa Ultrafree® −15 CentrigugalFilter Device (Millipore). Half of the purified Bo1 mAb was biotinylatedwith NHS-LC-biotin (Pierce) according to the manufacturer'srecommendations. Maxisorp™ microtitre plates (Nunc) were coated withunbiotinylated Bo1 mAb in PBS (2 μg/ml) for 2 hours at room temperature,washed 3 times with wash buffer (150 mM NaCl, 0.05% Tween 20 in 10 mMphosphate buffer, pH 7.2), and blocked with blotto (10 mM phosphatebuffer, containing 0.5% Tween 20, pH 7.2) and 5% bovine skim milkpowder. Following a further 6 washes, undiluted extracts from the woolsamples, positive controls (crude soluble antigen from whole lice inPBS) and negative controls (PBS plus 0.5% Tween 20) were then added tothe plates in duplicate for 1 hour at room temperature. Plates werewashed again 6 times and biotinylated Bo1 mAb (2 μg/ml) added for 1 hourat room temperature. Following 6 washes, strepavidin-horseradishperoxidase conjugate (2 μg/ml) was added for 1 hour. The enzyme reactionwas developed using tetramethylbenzidine substrate, the reaction wasstopped with 1 M sulphuric acid and adsorbance of the wells read at 450nm wavelength.

[0154] All 12 louse-naive lambs had zero louse scores and were negativein the ELISA (FIG. 6). Results from the 29 louse-infested lambs weresignificantly correlated with the louse scores (r=0.77, P<0.001, FIG.6). This assay can be used to detect louse infestation in sheep flocksand thereby assist farmers in the rational use of anti-louse treatmentswith consequent reduction of chemical residues in products from sheepand in the environment.

EXAMPLE 12 Diagnosis of Immunological Hypersensitivity to B. ovis invivo by Intradermal Skin Testing Using Purified Bo1 Antigen

[0155] Three louse-naive lambs and three louse-infested lambs, 12 monthsof age were used. The lambs were prepared for intradermal skin testingby closely shearing the wool from the upper shoulder region. The antigenand control solutions were injected intradermally in volumes of 0.1 ml.The diameters of the skin reactions were measured at 0.5, 5, 24, and 48hours after injection. Bo1 was purified as described in Example 5. TheBo1 in neutralised elution buffer was diluted to approximately 6.0 μgper ml (determined by absorbance at 280 nm) with PBS. Neutralisedelution buffer similarly diluted with PBS was the negative controlsolution for the Bo1. Crude soluble antigen prepared from whole B. ovisas described in Example 1 was diluted to 100 μg per ml. The negativecontrol for crude B. ovis antigen was PBS mixed 1:1 with glycerol andsimilarly diluted. Histamine HCl (1 in 250000, w/v) in PBS was used totest for responsiveness to histamine.

[0156] The skin test results show substantial responses to crude B. ovisantigen and Bo1 only in the louse-infested sheep (FIG. 7). The responseswithin 30 minutes were typical of hypersensitivity due to reaginic(typically IgE) antibody-mediated mechanisms while responses extendingto 5 hours and beyond indicate cellular mechanisms.

EXAMPLE 13 Use of Bo1 Antigen in an in vitro Assay to DetectImmunological Hypersensitivity to B. ovis.

[0157] An ELISA or similar assay may be used to detect IgE specific forBo1 in sheep sera as an alternative to intradermal skin testing todiagnose hypersensitivity to B. ovis. Alternatively, similar assays canbe used to detect other ovine immunoglobulin isotypes with specificityto Bo1 providing evidence of exposure of the host to the parasite.

[0158] To detect Bo1 specific IgE individual serum samples from sheepwere treated with SAS (saturated ammonium sulphate solution) to reducelevels of competing IgG. 70% SAS in saline was added in a 1 to 1 ratio(v/v) to the serum samples for 30 minutes with periodic mixing. Theprecipitate was spun down (13 000 g, 10 minutes) and the supernatantdiluted 1 to 8 in distilled water with 0.1% Tween 20 to give a finaldilution of 1 in 16. Standard ELISA procedures were followed and arebriefly described. Ninety-six well microtitre plates were coated withaffinity purified Bo1 (1 in 100 in PBS) for 5 hours at room temperature.The plates were blocked with 10 mM phosphate buffer, pH 7.2, containing0.5% Tween 20 and 5% bovine skim milk powder for 1 hour and then washed6 times with wash buffer (150 mM NaCl, 0.05% Tween 20 in 10 mM phosphatebuffer, pH 7.2). The SAS treated serums were added to duplicate wellsand held at room temperature for 1 hour and then at 4° C. overnight. Theplates were then washed 6 times with wash buffer and anti-ovine IgEmonoclonal antibody in dilution buffer (5 mg/ml BSA, 0.1% Tween 20 inPBS) was added for 4 hours at room temperature. Following washing, goatanti-mouse IgG conjugated to horseradish peroxidase (Sigma A3673, 1 in1000 in dilution buffer) was added for 1 hour at 37° C. The plates werewashed again and tetramethylbenzidine (0.1 mg/ml, Sigma T8768) in 0.1Macetate buffer pH 6.5 plus 0.1% DMSO and 0.03% hydrogen peroxide addedfor 15 minutes. The reactions were stopped with 1 M sulphuric acid andabsorbance at 450 nm read on an automated plate reader. The results fromthe ELISA using Bo1 were compared to a similar ELISA using crude B. ovisantigen (FIG. 8).

[0159] The ELISAs using whole louse antigen and purified allergen-coatedplates showed significantly higher reactivity of serum IgE from lousysheep than from louse-naive sheep (P≦0.0001 and 0.0025 respectively,FIG. 8). Sixty-three percent ({fraction (19/30)}) of the lousy sheepshowed B. ovis-specific IgE absorbances greater than the mean plus 2standard deviations of absorbances obtained using sera from louse-naivelambs (FIG. 8). The sera of 50% ({fraction (15/30)}) of thelouse-infested sheep showed elevated IgE absorbance to purified Bo1allergen based on the same criteria. This data indicates that thepurified allergen is a major allergen of B. ovis. In 13 of these sera,elevated IgE responses to both antigen preparations, were detected (FIG.8). However, within the lousy sheep, only moderate correlation betweenthe IgE responses to both antigen preparations was observed (r=0.49,P>0.006), suggesting the presence of other allergens in crude louseantigen and/or less non-specific reactivity when the purified Bo1allergen is used.

DISCUSSION

[0160] The applicants have demonstrated serum antibody responses(including IgG and IgE) specific for antigens of the louse B. ovis andspecific proliferation of lymphocytes obtained from skin draining lymphnodes in response to challenge with louse B. ovis antigens in vitro forthe first time. Additionally, immunological responses to the louse havebeen demonstrated by intradermal skin testing where responses within onehour largely reflect the presence of reaginic antibodies (IgE and otherhomocytotropic antibodies) and persistence of responses to 24 hours andbeyond largely reflect cell mediated mechanisms. The specificity ofthese various immunological responses was determined by comparing theresponses in louse-infested lambs to those in louse-naive lambsmaintained under similar conditions. The nature of the immunologicalresponses to the louse and the character of the cockle lesions asdetermined by histology supports the concept that Cockle, thesuperficial dermatitis leading to pelt defects in louse-infested sheep,is the consequence of an allergic (or hypersensitive) immunologicalresponse to the louse. The novel allergen of the present invention andligands binding to it may be used in diagnosis, prevention and treatmentof B. ovis infection and the associated allergic disease resulting inimproved wool and pelt quality, decreased use of synthetic insecticidesand improved economic gains for the farmer.

[0161] It should be appreciated that throughout this specificationwherever the term “comprises” (or grammatical variants thereof) is used,this term is not intended to be limiting and it does not exclude thepresence of other features or elements in the present invention. Thus,the word “comprises” is equivalent to the word “includes”.

[0162] Aspects of the present invention have been described by way ofexample only, and it should be appreciated that modifications andadditions may be made thereto without departing from the scope thereofas defined in the appended claims.

REFERENCES

[0163] Bany J, Pfeffer A and Phegan M (1995). Comparison of local andsystemic responsiveness of lymphocytes in vitro to Bovicola ovis antigenand Concanavalin A in B. ovis-infested and naive lambs. InternationalJournal for Parasitology 25:1499-1504.

[0164] Bany J, Pfeffer A, Phegan M and Heath A C G (1995). Proliferativeresponses of lymphocytes in Bovicola ovis-infested lambs. InternationalJournal for Parasitology. 25:765-768.

[0165] Cleland P C, Dobson K J and Meade R J (1989). Rate of spread ofsheep lice (Damalinia ovis) and their effects on wool quality.Australian Veterinary Journal. 66: 298-299.

[0166] Heath A C G, Cole D J W, Bishop D M, Pfeffer A T, Cooper S M, andRisdon P (1995). Preliminary investigations into the aetiology andtreatment of Cockle, a sheep pelt defect. Veterinary Parasitology.56:239-254.

[0167] Heath A C G, Cooper S M, Cole D J W and Bishop D M (1995).Evidence for the role of the sheep biting-louse Bovicola ovis inproducing cockle, a sheep pelt defect. Veterinary Parasitology.59:53-58.

[0168] Johnson P W, Boray J C, Plant J W and Blunt S C (1993).Prevalence of the causes of fleece derangement among sheep flocks in NewSouth Wales. Australian Veterinary Journal 70:220-224.

[0169] Kettle P R and Lukies J M (1982). Effects of sheep lice(Damalinia ovis) on wool colour. New Zealand Journal of ExperimentalAgriculture. 10:15-17.

[0170] Kettle P R and Lukies J M (1984). Recovery of sheep lice(Damalinia ovis) from baled wool: a technique enabling nation-widesurveillance of louse ridden flocks. New Zealand Journal of ExperimentalAgriculture 12:39-42.

[0171] Lipson M and Bacon-Hall R E (1976). Some effects of variousparasite populations in sheep on the processing performance of wool.Wool Technology and Sheep Breeding. pp18-20.

[0172] McLeod R S (1995). ‘Costs of major parasites to the Australianlivestock industries’. Proceedings of the Australian Society forParasitology Annual Meeting, 1994. In the International Journal forParasitology 25:1363-1367.

[0173] Pfeffer A T, Bany J, Phegan M D and Osborn P J (1993).‘Hypersensitivity skin testing of lambs infested with the biting louse(Bovicola ovis)’. Proceedings of the 23rd Conference of the NZ Societyfor Veterinary and Comparative Pathology, November 1993. In New ZealandVeterinary Journal 42:76.

[0174] Pfeffer A, Phegan M D and Bany J (1997). Detection ofhomocytotropic antibody in lambs infested with the louse, Bovicola ovis,using a basophil histamine-release assay. Veterinary Immunology andImmunopathology 57:315-325.

[0175] Seymour-Jones A (1913) ‘“Cockle” in Sheepskins’. In The sheep andIt's Skin, Seymour-Jones A. Leather Trades Review, London. Chapter VII,pp 204-221.

1 16 1 911 DNA Bovicola ovis misc_feature (1)...(911) n = A,T,C or G 1atcaaaacaa caatgcaagg attaaaatta attttcgtcg cctttttggc agttttcgct 60gttgggtgtg agggaaatac tttggtcaaa tccccaacag aactcgatct tcgtcttctt 120gttgaaaccg ctcgagatat ctctgtcatc ttgtttaaaa acttacatgc tggatataat 180gaagttaacc ccaaaatcga aatactgttg aacgaattgg cccccgaagc taaagaagga 240ctccaaaaaa ttataaaaga aattagagat ttggtcaatg aagaagaaac cagaattaat 300gtcatcttca aaactcttat tggtgctttg gaccaactga aaccaattaa ggcaccatgc 360gccgaccccg tttctaaaga agctaaaaaa ttggccaacg atgttgaaag ggaaatcgtc 420aaattcatta aatatttaga acaaaaatac gaaaaggtat ttacaaacat caagaatgga 480gttaccaaag taatcaccag agccaggaaa ttgtttgaca ctgaagttcc cgaagtcgtg 540aaatgtttga cccccaaaaa caaagaggcc actaaatgca tcaatacaca catcgacaaa 600attcttggtg aagttgccca aatcggtgcc gacattggac tccttgtaat ctcttctgaa 660gaagctctta atcccgttat taaggaagtt gtcgccaaaa taggtgaaca agtgttgaag 720gttttgggtg aaggtaggcc cattatcaac aaaatctcag actgtgttgc aaaaatgtaa 780gaaataaaaa gaaataagtn aataaattaa ttttaatttt tttttaattt tttttttctt 840taatgccaaa caaaaaaatt aaaaattttt aaatnaattt taaaaattaa aaaaaaaaaa 900aaaaaaaaaa a 911 2 254 PRT Bovicola bovis 2 Met Gln Gly Leu Lys Leu PheVal Ala Phe Leu Ala Val Phe Ala Val 1 5 10 15 Gly Cys Glu Gly Asn ThrLeu Val Lys Ser Pro Thr Glu Leu Asp Leu 20 25 30 Arg Leu Leu Val Glu ThrAla Arg Asp Ile Ser Val Ile Leu Phe Lys 35 40 45 Asn Leu His Ala Gly TyrAsn Glu Val Asn Pro Lys Ile Glu Ile Leu 50 55 60 Leu Asn Glu Leu Ala ProGlu Ala Lys Glu Gly Leu Gln Lys Ile Ile 65 70 75 80 Lys Glu Ile Arg AspLeu Val Asn Glu Glu Glu Thr Arg Ile Asn Val 85 90 95 Ile Phe Lys Thr LeuIle Gly Ala Leu Asp Gln Leu Lys Pro Ile Lys 100 105 110 Ala Pro Cys AlaAsp Pro Val Ser Lys Glu Ala Lys Lys Leu Ala Asn 115 120 125 Asp Val GluArg Glu Ile Val Lys Phe Ile Lys Tyr Leu Glu Gln Lys 130 135 140 Tyr GluLys Val Phe Thr Asn Ile Lys Asn Gly Val Thr Lys Val Ile 145 150 155 160Thr Arg Ala Arg Lys Leu Phe Asp Thr Glu Val Pro Glu Val Val Lys 165 170175 Cys Leu Thr Pro Lys Asn Lys Glu Ala Thr Lys Cys Ile Asn Thr His 180185 190 Ile Asp Lys Ile Leu Gly Glu Val Ala Gln Ile Gly Ala Asp Ile Gly195 200 205 Leu Leu Val Ile Ser Ser Glu Glu Ala Leu Asn Pro Val Ile LysGlu 210 215 220 Val Val Ala Lys Ile Gly Glu Gln Val Leu Lys Val Leu GlyGlu Gly 225 230 235 240 Arg Pro Ile Ile Asn Lys Ile Ser Asp Cys Val AlaLys Met 245 250 3 30 PRT Artificial Sequence N-terminal aa sequence 3Ser Pro Thr Glu Leu Asp Leu Arg Leu Leu Val Glu Thr Ala Arg Asp 1 5 1015 Ile Ser Val Ile Leu Phe Lys Asn Leu His Ala Gly Tyr Asn 20 25 30 4 8PRT Artificial Sequence artificially synthesized peptide 4 Asp Ile SerVal Ile Leu Phe Lys 1 5 5 12 PRT Artificial Sequence artificiallysynthesized peptide 5 Asn Leu His Ala Gly Tyr Asn Glu Val Asn Pro Lys 15 10 6 6 PRT Artificial Sequence artificially synthesized peptide 6 ValPhe Thr Asn Ile Lys 1 5 7 7 PRT Artificial Sequence artificiallysynthesized peptide 7 Ile Gly Glu Gln Val Leu Lys 1 5 8 6 PRT ArtificialSequence artificially synthesized peptide 8 Ile Asn Val Ile Phe Lys 1 59 12 PRT Artificial Sequence artificially synthesized peptide 9 Lys LeuPhe Asp Thr Glu Val Pro Glu Val Val Lys 1 5 10 10 8 PRT ArtificialSequence artificially synthesized peptide 10 Asp Ile Ser Val Ile Leu PheLys 1 5 11 13 PRT Artificial Sequence artificially synthesized peptide11 Ile Glu Ile Leu Leu Asn Glu Leu Ala Pro Glu Ala Lys 1 5 10 12 10 PRTArtificial Sequence artificially synthesized peptide 12 Thr Leu Ile GlyAla Leu Asp Gln Leu Lys 1 5 10 13 26 DNA Artificial Sequence BoP14-Aoligonucleotide 13 catgctggat ataatgaagt waaycc 26 14 29 DNA ArtificialSequence BoP14-B oligonucleotide 14 ttaacaactt caggaacttc wgtrtcraa 2915 31 DNA Artificial Sequence Bo1-X2 primer 15 cttgcggccg ccatttttgcaacacagtct g 31 16 32 DNA Artificial Sequence Bo1-X3 primer 16cgcggatcca tatgtcccca acagaactcg at 32

What is claimed is:
 1. A substantially purified polypeptide which hasthe amino acid sequence of SEQ ID NO. 2, or a fragment or a variant ofsaid polypeptide having substantially equivalent activity.
 2. Thepolypeptide of claim 1, wherein said polypeptide is derived from a lousewhich provokes a humoral and/or cellular immunological response in ananimal infested by the louse.
 3. The polypeptide of claim 2 wherein thevariant or fragment incorporates a B cell or a T cell epitope of thepolypeptide. 4-8. (Canceled)
 9. An isolated nucleic acid moleculeencoding a polypeptide selected from the group consisting of: a) thepolypeptide according to claim 1; b) the polypeptide of (a) abovewherein the polypeptide is derived from a louse which provokes a humoraland/or cellular immunological response in an animal infested by thelouse, or a fragment or variant of said polypeptide having substantiallyequivalent activity thereto; and c) the polypeptide of (b) above whereinthe fragment or variant of said polypeptide incorporates a B cell or a Tcell epitope of the polypeptide.
 10. An isolated nucleic acid moleculewherein the molecule: a) comprises a nucleotide sequence of SEQ ID NO.1; or b) is a functional fragment or variant of the molecule in (a)above; or c) is able to hybridize under stringent conditions to themolecule in (a) above; or d) is the complement of the molecule definedin (a), (b) or (c) above; or e) is an anti-sense sequence correspondingto any of the sequences in (a)-(d) above.
 11. (Canceled)
 12. The vectorcomprising the nucleic acid molecule of claim
 10. 13. A host cell whichhas been transformed with a vector of claim
 12. 14. A ligand which bindsto the polypeptide of claim
 1. 15. The ligand of claim 14 wherein theligand is an antibody or a fragment of an antibody containing thebinding domain.
 16. The ligand of claim 14 wherein the ligand is amonoclonal antibody.
 17. The ligand of claim 14 wherein the ligand is aphage display molecule.
 18. A method for assaying samples for thepresence of ligands which bind to Bo1 or a segment thereof comprisingobtaining an excretion, secretion, tissue or blood sample from the hostand exposing the sample to a Bo1 ligand binding agent or Bo1 probe by anELISA or other suitable assay.
 19. The method of claim 18 wherein thesample to be assayed is first prepared as a substrate solution.
 20. Atest kit suitable for use in the assay of claim 18 wherein the kitcomprises a Bo1 ligand binding agent or probe incorporated into an ELISAor other suitable assay.
 21. An isolated probe capable of hybridizingunder stringent conditions to the nucleic acid molecule of claim
 10. 22.A method of diagnosing hypersensitivity to Bovicola ovis or Bo1polypeptide in an animal comprising: a) injecting intradermally at asite a suitable amount of the polypeptide of claim 1 together with apharmaceutically or veterinarily suitable carrier or diluent; b)examining at appropriate times thereafter the site to observe a reactionto the polypeptide; and c) comparing these observations with thoseproduced by injections of control solutions to determine whether ahypersensitivity to the polypeptide was present.
 23. A vaccine toprevent or reduce Bo1 hypersensitivity in susceptible animals whereinthe vaccine comprising an agent selected from the group consisting of:a) a substantially purified polypeptide which has an amino acid sequenceof SEQ ID NO. 2, or a fragment or a variant of said polypeptide havingsubstantially equivalent activity thereto; b) the nucleic acid moleculeof claim 10; c) organisms transfected with and/or expressing a nucleicacid molecule encoding the polypeptide according to (a) above; and d)ligands or probes which bind to the polypeptide of (a) above.
 24. Amethod of treating animals or preventing animals from exhibitingallergic hypersensitivity to Bo1 polypeptide comprising the step ofadministering to said animal an effective amount of the vaccine of claim23.
 25. A composition including an ineffective amount of an agentselected from the group consisting of: a) the nucleic acid of claim 10;b) a substantially purified polypeptide which has an amino acid sequenceof SEQ ID NO. 2, or a fragment or a variant of said polypeptide havingsubstantially equivalent activity thereto; c) organisms transfected withand/or expressing a nucleic acid molecule encoding the polypeptide of(b) above; and d) ligands or probes which bind to the polypeptide of (b)above; together with a pharmaceutically or veterinarily suitable carrieror diluent.
 26. The composition of claim 25 wherein the compositionfurther comprises at least one adjuvant or cytokine.
 27. A method oftreating animals or preventing animals from exhibiting allergichypersensitivity to the Bo1 polypeptide comprising administering aneffective amount of the composition of claim 25 to said animal. 28-29.(Canceled)
 30. A method of diagnosing ectoparasite infestationcomprising obtaining an excretion, secretion, tissue or blood samplefrom the host; and exposing the sample to a ligand or probe for anidentified antigen present in the ectoparasite's faeces by an ELISA orother suitable assay. 31-32. (Canceled)
 33. A test kit for diagnosingectoparasite infestation by the method of claim 30 wherein the kitcomprises a ligand or probe for an identified antigen present in theectoparasite's faeces incorporated into an ELISA or other suitableassay.
 34. The test kit as claimed in claim 33 wherein the ectoparasiteis B. ovis and wherein the test kit comprises a ligand for Bo1incorporated into an ELISA or other suitable assay.